Endocrine abnormalities occur either as ?self-defined? disorders or in combination with other diseases. An ever-increasing number of ?non-endocrine? diseases also are found to be associated with endocrine abnormalities. Optimal use of valid laboratory tests and correct diagnosis of endocrine diseases or the endocrine components of ?non-endocrine? diseases obviously are important both medically and economically. Thyroid diseases represent the most common endocrine abnormalities, while other endocrine diseases such as pheochromocytoma are relatively uncommon. Although pheochromocytoma is a rare cause of hypertension, case findings require screening of a large numbers of patients. Nearly one in four Americans has hypertension, but only 0.1-0.5% of these patients will be found to have pheochromocytoma. Nevertheless, diagnosing these patients is important since ~90% can be cured with surgery, while the consequences can be catastrophic for the patient if the disease goes unrecognized and/or untreated. Despite the increasing use of catecholamine and metanephrine measurements in clinical laboratories for the diagnosis of pheochromocytoma (and for a variety of other conditions/diseases), information regarding optimal specimen collection remains limited. We compared the two most commonly used anticoagulants on catecholamine and metanephrine measurements. Heparin- and EDTA-anticoagulated venous blood (without added antioxidant[s]) was obtained from healthy subjects and patients who were evaluated for pheochromocytoma. Plasma was separated from cells within 1 to 3 hours after collection. Plasma specimens were frozen from 1 day to 14 months at -70 Celsius degrees until testing. Plasma catechols (L-dopa, dopamine, norepinephrine, epinephrine, 3,4-dihydroxyphenylacetic acid [DOPAC], 3,4-dihydroxyphenylglycol [DHPG]) and free (unconjugated) methoxytyramine and metanephrines (normetanephrine, metanephrine) were measured by high-performance liquid chromatography (HPLC) methods as described previously. Although the differences did not always reach statistical significance (paired t-test), the concentrations of various catecholamines and catecholamine metabolites were all lower in EDTA plasma than heparin plasma. In contrast, the concentrations of free methoxytyramine and free metanephrines tended to be higher in EDTA than heparin plasma, but the differences were not statistically significant. Our findings show that the choice of anticoagulant (heparin vs. EDTA) has clinically important effects on plasma catecholamine measurements and also may affect measurements of free methoxytyramine and metanephrines. We interpret the data as indicating that plasma catechols are less stable in EDTA blood than heparinized blood. The reciprocal differences in O-methylated metabolites suggest involvement of the red blood cell enzyme catechol-O-methyltransferase (COMT). The practical implications of our observations are two-fold. First, since EDTA appears to be the most widely used anticoagulant for the measurements of catechols and metanephrines, laboratories may need to re-assess its use for specimen collection. Second, observed differences in reference intervals for these analytes between various laboratories may, at least in part, attributable to the choice of anticoagulants and this needs to be recognized for correct interpretation of the test results. In a collaborative study, we showed that measurements of plasma free metanephrines not only provide information about the likely presence or absence of a pheochromocytoma, but when a tumor is present, can also help predict tumor size and location. This additional information may be useful for clinical decision-making during tumor localization procedures. While measurements of plasma-free normetanephrine and metanephrine provide a sensitive test for diagnosis of pheochromocytoma, these tests may fail to detect tumors that produce predominantly dopamine. Such tumors are extremely rare, usually found as extraadrenal paragangliomas. In a collaborative study we showed that measurements of plasma free methoxytyramine, in addition to normetanephrine and metanephrine, are unlikely to improve diagnosis of pheochromocytomas in hypertensive patients with symptoms of catecholamine excess but may be useful in selected patients for identification of tumors that produce predominantly dopamine.